Combining LOPIT with differential ultracentrifugation for high-resolution spatial proteomics.


Type
Article
Change log
Authors
Geladaki, Aikaterini  ORCID logo  https://orcid.org/0000-0002-0530-4252
Kočevar Britovšek, Nina 
Breckels, Lisa M 
Abstract

The study of protein localisation has greatly benefited from high-throughput methods utilising cellular fractionation and proteomic profiling. Hyperplexed Localisation of Organelle Proteins by Isotope Tagging (hyperLOPIT) is a well-established method in this area. It achieves high-resolution separation of organelles and subcellular compartments but is relatively time- and resource-intensive. As a simpler alternative, we here develop Localisation of Organelle Proteins by Isotope Tagging after Differential ultraCentrifugation (LOPIT-DC) and compare this method to the density gradient-based hyperLOPIT approach. We confirm that high-resolution maps can be obtained using differential centrifugation down to the suborganellar and protein complex level. HyperLOPIT and LOPIT-DC yield highly similar results, facilitating the identification of isoform-specific localisations and high-confidence localisation assignment for proteins in suborganellar structures, protein complexes and signalling pathways. By combining both approaches, we present a comprehensive high-resolution dataset of human protein localisations and deliver a flexible set of protocols for subcellular proteomics.

Description
Keywords
Cell Fractionation, Cell Line, Tumor, Centrifugation, Density Gradient, Humans, Mass Spectrometry, Proteome, Proteomics, Spatial Analysis, Ultracentrifugation
Journal Title
Nat Commun
Conference Name
Journal ISSN
2041-1723
2041-1723
Volume Title
10
Publisher
Springer Science and Business Media LLC
Sponsorship
Biotechnology and Biological Sciences Research Council (BB/N023129/1)
Wellcome Trust (110170/Z/15/Z)
Biotechnology and Biological Sciences Research Council (BB/K00137X/1)
Wellcome Trust (108467/Z/15/Z)
Biotechnology and Biological Sciences Research Council (BB/R505365/1)
BBSRC (1947751)
Wellcome Trust BBSRC